A variety of vascular grafts are commercially available and include mechanical, bioprosthetic, and cryopreserved and decellularized human and animal heart valves. In addition, non-valved vascular grafts made of expanded polytetrafluoroethylene (ePTFE), decellularized veins and arteries, and cryopreserved veins and arteries are available. Vascular grafts constructed from naturally occurring molecules, for example collagens, elastins, hyaluronins, etc. can be manufactured using tissue engineering techniques. Such vascular grafts, whether valved or non-valved can be used in clinical applications in an essentially cellularized on decellularized state. The current invention is directed at providing a device and process for recellularizing essentially acellular, i.e. avital, vascular grafts derived from human or animal sources or as constructed using any number of tissue engineering methodologies.
Vascular grafts include a wide variety of natural and synthetic tubular structures that may or may not contain valves. Valves in these tubular structures are usually intended to direct the flow of blood (or other nutrient materials) in one direction by preventing the backward flow of this (these) liquid solution(s). Examples of valved tubular structures include aortic, pulmonary, and mitral valves present in the hearts of most vertebrate animals and veins used to return blood flow from the periphery of the body to the heart for recirculation. Vascular grafts constructed of synthetic materials include devices constructed from man-made polymers, notably Dacron and Teflon in both knitted and woven configurations such as those marketed by W. L. Gore, Inc. and Impra, Inc. where various forms of polytetrafluoroethylene (PTFE) are molded into a wide array of tubule structures (see for example U.S. Pat. Nos. 4,313,231; 4,927,676; and 4,655,769). The present invention does not involve vascular grafts derived from synthetic means and thus these types of vascular grafts will not be further discussed. Natural vascular grafts, taken in the context of this present invention, include valved and non-valved tubular structures obtained by methodologies broadly classified under the term xe2x80x9ctissue engineeringxe2x80x9d. Notably, tissue engineered blood vessels such as described in U.S. Pat. Nos. 4,539,716, 4,546,500, 4,835,102, and blood vessels derived from animal or human donors such as described in U.S. Pat. Nos. 4,776,853, 5,558,875, 5,855,617, 5,843,181, and 5,843,180, and a pending patent application entitled xe2x80x9cA Production Technology for Commercial Scale Decellularization Processing of Soft-Tissue Engineered Medical Implantsxe2x80x9d (patent application Ser. No. 09/528,371 incorporated herein in its entirety) are known. The present invention involves vascular grafts derived using a specific process associated with tissue engineering as well as a bioreactor device to be used in this process.
Tissue engineered natural vascular grafts, hereinafter vascular grafts, can be manufactured by processing of natural vascular grafts (veins, arteries, heart valves, etc.) with the objective of removing the cellular elements without damaging the matrix structure of that tissue-a xe2x80x9creductionistxe2x80x9d approach. This approach is generally referred to as decellularization and is the subject of several patents, of which U.S. Pat. No. 4,801,299 by Brendel and Duhamel is considered as one of the earliest such patents, and pending patent applications as described above. Decellularization of tissues such as vascular grafts can be readily accomplished by incubating tissues in the presence of detergents, both anionic and nonionic, with and without digestion of nucleic acids using DNase and RNase enzymes, or more recently a commercially available recombinant endonuclease called Benzonase(trademark). The decellularized tissues typically retain the structurally important molecules such as collagens, elastins, proteoglycans, and associated polysaccharides such as the hyaluronins, (see U.S. Pat. No. 5,855,620 as an example). Specifically, the Brendel and Duhamel patent (U.S. Pat. No. 4,801,299) defines decellularization as xe2x80x9cA method of treating body tissue to remove cellular membranes, nucleic acids, lipids, and cytoplasmic components and form extracellular matrix having as one major component collagens and making said body tissue suitable for use as a body implant . . . xe2x80x9d The Klement, Wilson, and Yeger patent (U.S. Pat. No 4,776,853) defines decellularization as xe2x80x9cA process for preparing biological material for implant in a mammal""s cardiovascular system, respiratory system or soft tissue by removing cellular membranes, nucleic acids, lipids, and cytoplasmic components and forming an extracellular matrix having as major components collagens and elastins . . . xe2x80x9d A devitalization process/method (i.e. producing an avital tissue) would be defined for purposes of the present invention as a process or method of treating body tissue to remove cellular membranes, nucleic acids, lipids, and small molecular weight cytoplasmic components forming an extracellular matrix having as one major component collagens, elastins, and high molecular weight polysaccharides. By leaving the high molecular weight cytoplasmic molecules, for example actins, complete decellularization would not be obtained and the tissue would be considered to be devitalized. In specific instances, patented technologies have suggested that such decellularized vascular grafts can function for extended periods of time following clinical implantation without the need for recellularization. Indeed some such technologies have treated tissues with high concentrations of the anionic detergent sodium docecylsulfate (SDS) to attempt to prevent recellularization of implanted vascular grafts (U.S. Pat. Nos 4,776,853 and 5,558,875).
Tissue engineered natural vascular grafts have also been constructed using a xe2x80x9cconstructionistxe2x80x9d approach. This approach involves the extraction of natural cellular and matrix components to obtain purified (or partially purified) fractions and then using these fractions to reconstruct a vascular graft from individual components. Alternatively, specific components of a vascular graft, for example collagen(s), can be obtained using recombinant DNA technologies and such highly purified and homogeneous materials used in the construction of natural vascular grafts via tissue engineering. Methods and materials for 3-dimensional cultures of mammalian cells are known in the art. See, e.g., U.S. Pat. No. 5,266,480. Typically, a scaffold is used in a bioreactor growth chamber to support a 3-dimensional culture, see for example U.S. Pat. No. 6,008,049. The scaffold can be made of any porous, tissue culture compatable material(s) into which cultured mammalian cells can enter and attach.
Both the reductionist and constructionist approaches are designed to provide an acellular matrix (unless the constructionist approach also intends to incorporated living cells in the matrix during the construction of the tissue engineered vascular graft) that can be used directly as an acellular graft. Alternatively the acellular matrix can be reseeded with specific cell populations to provide a recellularized graft (see for example, U.S. Pat. Nos. 5,792,603; 5,613,982; 5,855,617; 5,843,180; 5,843,181; and 5,843,182). One U.S. Pat. No. 5,855,617 describes the use of fibroblast growth factor to attract fibroblast cells to migrate into a substantially non-immunogenic vascular graft.
Vascular tissues such as an aortic heart valve contain a limited number of cell types. For aortic valves, these cell types include endothelial cells that line the luminal surface of the valve providing for a smooth, non-thrombogenic, surface for efficient blood flow. These cells are known for their role in nitric oxide (a vasodilating agent), expression of vasoconstricting endothelins, and smooth muscle proliferative mitogens and cytokine (Interleukin-1, tumor necrosis factor, Interleukin 6, Interleukin 8, Monocyte Chemoattractant protein-1, granulocyte monocyte-CSF, and Monocyte chemoattractant and stimulating factor) production. These endothelial cells are attached to a thin basement membrane that is comprised essentially of high density, i.e. minimal porosity, type IV collagens. The basement membrane is visible, following removal of endothelial cells, as a smooth surface essentially devoid of breaks in integrity and closely following the topography of the underlying vascular structure. Within the underlying vascular structure, the cellular elements primarily consist of a fibroblastic cell population, typically referred to as myofibroblasts in that their origin is described as being muscle and specifically cardiac muscle and smooth muscle cells. This fibroblastic cell population has been described as being an important element to the continued repair and synthesis of the molecular elements comprising the heart valve matrix. Of relevance to this present invention are observations such as:
1) The basement membrane lining the luminal surface of a vascular graft is essentially impenetrable by cells due to its high density of structural elements, i.e. minimal porosity to cells due to small openings in the matrix structure;
2) The small dimensions of pores present in the matrix structure of the basement membrane are too small for cells to easily penetrate;
3) Type IV collagen has been described as being inhibitory to cellular proliferation in in vitro, such studies suggesting a strong potential to chemically inhibit trans-membrane migration of cells;
4) Endothelial cells and matrix fibroblasts communicate via a paracrine (local) chemical signaling process, where this signaling is important to the continued function and well-being of both cell populations;
5) Transplanted, cryopreserved, human heart valves containing viable fibroblastic cell populations at the time of transplantation into a patient have been reported to be devoid of viable fibroblastic cells in as little as a few months post transplantation, supportive of the concept of an important paracrine signaling function between cells of a native valve and an inductive process of apoptosis;
6) Transplanted, cryopreserved, human heart valves continue to function for as long as 20 years post transplantation in spite of the loss of both the endothelial and fibroblastic cell populations, suggesting that the absence of a viable fibroblastic cell population may be of minimal importance to long-term function of an aortic valve;
7) Transplanted, cryopreserved, human heart valves recellularize only poorly by recipient fibroblastic cells post transplantation, suggesting an inherently poor inducement by the transplanted tissue for cells to migrate into the matrix structure of a cardiovascular graft; and
8) Transplanted, cryopreserved, human heart valves reendothelialize only poorly by recipient endothelial cells post transplantation and where reendothelialization occurs, it is primarily at the surgical anastomoses.
Collectively, these published observations tend to suggest that simple incubation of fibroblastic cells with cardiovascular tissues will result in successful cellular penetration into the matrix structure only via the adventitial (outside) surface of the vascular graft. Such penetration will most likely require some inducement other than simply incubating cells with this surface hoping the cells will attach to the adventitial surface and migrate into the tissue. Penetration of fibroblastic cells into the tissue matrix via the luminal surface will require partial or complete removal of the basement membrane, or strategic perforation of the basement membrane. In that cardiovascular tissues where the basement membrane remain intact mostly fail to recellularize by recipient cells post-transplantation would seem to suggest that recellularization without inducement is an unlikely event. In addition, the high flow-fields (high rates of blood flow through an aortic valve) or an inherent inability of endothelial cells to migrate along the surface of the basement membrane tends to suggest that reendothelialization of a vascular graft will require more than simple incubation of endothelial cells in suspension cultures with a vascular graft. Some effort will need to be made to attach and align the endothelial cells such that the flow of fluids across their surfaces will not tend to detach them until they have had an opportunity to align themselves post seeding. Such alignment will presumably require pulsatile fluid flow to mimic in situ conditions for endothelial cell function. U.S. Pat. No. 5,928,945 describes the tissue engineered production of cartilage where shear flow stress of about 1 to about 100 dynes/cm2 produce artificial cartilage when cells are grown in a bioreactor on an artificial substrate.
U.S. Pat. No. 5,792,603 (hereinafter xe2x80x9cpatent 603xe2x80x9d) entitled xe2x80x9cApparatus and method for sterilizing, seeding, culturing, storing, shipping and testing tissue, synthetic or native, vascular graftsxe2x80x9d describes a bioreactor mediated apparatus and method for sterilizing, seeding, culturing, storing, shipping, and testing vascular grafts. This patent differs from the present invention in several important aspects.
1) Patent 603 describes alternating pressure to a support structure within the treatment chamber upon which a vascular graft scaffold is positioned. The present invention describes alternating pressure to a vascular graft within the treatment chamber and there is no associated support structure.
2) Patent 603 describes applying a radial/shear stress on the scaffold where the associated vascular graft scaffold and associated formed vascular graft is/are attached at one end and discharges solution flow directly into the treatment chamber such that the pulsatile pressure is not contained within the lumen of the scaffold/vascular graft. The present invention describes applying a radial/shear stress on the vascular graft where the vascular graft is attached to the treatment chamber (bioreactor) such that solution flow induced radial stress is controllable by regulating pressure between the advential side of the vascular graft and the inner wall of the treatment chamber and pulsatile pressure is contained within the lumen of the vascular graft.
3) Patent 603 relies on cellular ingrowth into the formed vascular graft supported by the vascular graft scaffold from the nutrient solutions being pumped through the lumen of the formed vascular graft and into the treatment chamber prior to this solution plus cells exiting the treatment chamber for recirculation to the treatment chamber. The present invention relies on a pressure differential between the luminal side and adventitial side of the vascular graft, generated by closing the outflow port of the luminal volume of the graft and opening the outflow port of the treatment chamber (bioreactor) associated with the volume outside of the luminal volume of the vascular graft. This pressure differential causes cells to move with solution flow across the volume of the tissue, luminal surface to adventitial surface, comprising the vascular graft such that the cells become entrapped within the matrix volume of the vascular graft.
4) Patent 603 does not control the volume of the luminal volume of formed vascular grafts. The present invention utilizes an inflatable piston to control the volume of the luminal volume of vascular grafts.
5) Patent 603 generates radial and/or shear stresses within the formed vascular grafts without controlling the stress (for example: force per unit cross-sectional area) and strain (for example: deformation from the unstressed state) on the formed vascular graft. The present invention allows control of the stress and strain on the vascular graft during the recellularization and preconditioning stages of the process in that the graft is attached at both ends to inflow and outflow ports and graft deformation during pulsatile pressure mediated flow of solutions can be controlled by management of the volume of solution between the advential (out) side of the vascular graft and the inner wall of the treatment chamber.
Based on these differences, it is suggested that the present invention is dramatically different from Patent 603 in the manner in which cells are induced to repopulate the vascular graft and in radial and shear stresses used to induce the cells repopulating the graft to experience physiologically relevant mechanical forces. The present invention, by being able to control stress and strain values applied to the vascular graft permit a more physiological and mechanically relevant conditions within the recellularized vascular graft(s). The manner in which the vascular grafts are attached in the treatment chamber (bioreactor) are significantly different between Patent 603 and the present invention. In Patent 603 the graft is attached at one end whereas in the present invention, the graft is attached at both ends.
The present invention is designed to take advantage of knowledge gained from prior art without actually incorporating prior art into the invention. The present invention provides a bioreactor approach to reseeding of vascular grafts, such as a decellularized aortic heart valve. The approach involves removal of the basement membrane by enzymatic digestion. This removal of basement membrane is followed by pressure-differential induced movement of fibroblastic cells in a solution into the matrix structure and reendothelialization by incorporation of endothelial cells into a collagenous/noncollagenous solution. This latter solution is compacted, as necessary, by xe2x80x9cpressurexe2x80x9d binding of this mixture onto the luminal surface to recreate a xe2x80x9cbasement membranexe2x80x9d containing endothelial cells. Cells are induced to resume metabolic activities following treatment with specific growth factors, for example fibroblast growth factor, or platelet aggregation under a pulsatile flow of nutrient solutions. The novel design of the bioreactor facilitates the processes described in the present invention.
Definitions
As used herein, xe2x80x9crecellularizationxe2x80x9d means the repopulation of the matrix volume of a tissue engineered or devitalized-essentially acellular/decellularized-acellular matrix structure with a viable cell population that is either the desired cell phenotype and/or genotype or that which can be caused to differentiate into the desired cell phenotype and/or genotype.
As used here in, xe2x80x9cnonvitalxe2x80x9d means tissue that has been treated to inactive the metabolic and/or reproductive capacity of cells residing within the tissue or residing on the luminal/adventialial surface(s) of the tissue(s). The cells are not necessarily disrupted and/or solubilized and may be visibly discernable using standard histological means and standard microscopic techniques known to persons skilled in the art. Alternatively, nonvital (or devitalized tissue) could mean tissue that has been treated to remove essentially all of the visible cellular remnants leaving only high molecular weight cytoplasmic moleculars such as, for example, actins.
As used herein, xe2x80x9creendothelialization means the repopulation of the flow surface of a tissue engineered or decellularized-acellular matrix structure with a metabolically and reproductively viable endothelial cell population that is either phenotypically and/or genotypically an endothelial cell at the time of repopulation or that which can be caused to differentiate into the desired cell phenotype and/or genotype.
As used herein, xe2x80x9cdecellularizationxe2x80x9d means the removal of cells and cell remnants from a tissue matrix structure using liquid solution processing such that cells are not visibly present, using standard microscopic techniques, in standard histologic preparations.
As used herein, xe2x80x9ccell to cell communicationxe2x80x9d means the chemical and/or physical signaling between one or more cells and/or cell populations such that a given cell or cell population is stimulated to function in a manner necessary to the role of that cell in maintaining tissue function. This cell to cell communication can occur by paracrine types of signaling using large and small molecular weight factors and is generally restricted to within the tissue comprising the functional entity being observed or studied.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art of tissue processing and cell culturing techniques. Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
According to one aspect of the present invention, a device and process is described for recellularizing and reendothelializing essentially accellular vascular grafts for use in replacement of defective heart valves and vascular conduits. The device is a bioreactor designed to facilitate selected steps in the processing such as recellularization and reendothelialization. The process includes several steps which may be conducted outside of the bioreactor and several steps which may be conducted inside of the bioreactor such that most of the invention is carried out in a closed processing system that will dramatically restrict contamination by microbiological and chemical/biological elements. In one preferred aspect, the process comprises the following steps:
1) use of an essentially acellular or non-vital vascular graft, such as a heart valve, whether constructed as an acellular graft using tissue engineering methods or decellularizing and/or devitalizing a native vascular graft using methods known in the art;
2) attaching the acellular/devitalized graft into the bioreactor by attaching the graft directly to the inlet and outlet port connections or by sewing/attaching the graft to sewing rings and attaching the sewing rings to the inlet and outlet port connections and closing the unit as illustrated in the attached figures;
3) optionally treating the acellular graft with various growth and/or differentiation factors, such as fibroblast growth factor (FGF), polylysine, hyaluronins, proteoglycans, RGD-containing peptides, sodium dodecylsulfate, sodium dodecylsarcosinate, or suramin, to achieve binding in the tissue matrix;
4) washing the acellular or devitalized graft with an appropriate aqueous solution to remove unbound, or loosely bound growth and/or differentiation factors;
5) debriding the basement membrane using proteolytic enzymes, for example dispase and/or collagenase, to achieve total or partial removal of the basement membrane lining the luminal surface of the acellular graft;
6) washing the acellular or devitalized graft with an appropriate aqueous solution to remove excess proteolytic enzymes;
7) seeding the acellular graft or devitalized graft with a fibroblastic cell population, allogenously or autogenously derived, using a positive pressure mediated infusion of cells into the tissue matrix spaces;
8) washing the recellularized graft with an appropriate iso-osmotic solution such that only the luminal volume and the volume outside of the vascular graft are replaced and no additional pressure flow occurs across the matrix of the recellularized graft;
9) seeding the recellularized graft with an endothelial cell population, allogenously or autogenously derived, using a viscous collagenous/noncollagenous mixture containing the endothelial cells;
10) partially pressurizing the luminal volume to compress the viscous collagenous/noncollagenous/endothelial cell mixture onto the luminal surface of the now recellularized and reendothelialized graft;
11) washing the now recellularized and reendothelialized graft to remove excess viscous collagenous mixture;
12) applying a slow pulsatile flow of nutrient rich and growth factor containing medium, optionally containing allogenous or autogenous platelets, to establish a functionally viable cell population in the graft such that the fibroblastic cell population and the endothelial cell population establish a viable vascular graft;
13) applying a pulsatile flow of nutrient rich medium at a flow rate appropriate to provide a stress field in the tissue graft appropriate to the physiological stimulation of the cell population in that tissue graft; and
14) preserving the now recellularized and reendothelialized graft by methods know in the art of cryopreservation, cold-storage preservation, and/or nutrient-culture preservation, or directly shipping and implanting the graft post recellularizing/reendothializing.